pharmaceutics
ArticleInhibition of the Hepatic Uptake of 99mTc-Tetrofosmin Using an
Organic Cation Transporter Blocker
Kodai Nishi 1, * , Masato Kobayashi 2 , Minori Kikuchi 3 , Asuka Mizutani 2 , Yuka Muranaka 2 , Ikumi Tamai 4 ,Keiichi Kawai 2,5 and Takashi Kudo 1
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Citation: Nishi, K.; Kobayashi, M.;
Kikuchi, M.; Mizutani, A.; Muranaka,Y.; Tamai, I.; Kawai, K.; Kudo, T.
Inhibition of the Hepatic Uptake of99m

Tc-Tetrofosmin Using an Organic

Cation Transporter Blocker.
Pharmaceutics 2021, 13, 1073.
https://doi.org/10.3390/
pharmaceutics13071073
Academic Editor: Björn Wängler
Received: 8 June 2021
Accepted: 8 July 2021
Published: 13 July 2021

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affil-

Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto,Nagasaki 852-8523, Japan; tkudo123@nagasaki-u.ac.jp
Faculty of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-1192, Japan; kobayasi@mhs.mp.kanazawa-u.ac.jp (M.K.);
mizutani.a@staff.kanazawa-u.ac.jp (A.M.); yukarisa93@stu.kanazawa-u.ac.jp (Y.M.);
kei@mhs.mp.kanazawa-u.ac.jp (K.K.)
School of Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; minolith04@gmail.com
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa
University, Kakuma, Kanazawa 920-1192, Japan; tamai@p.kanazawa-u.ac.jp
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji,
Fukui 910-1193, Japan
Correspondence: koudai@nagasaki-u.ac.jp; Tel.: +81-95-819-7103; Fax: +81-819-7104

Abstract: The accumulation of high levels of 99m Tc-tetrofosmin (99m Tc-TF) in the hepatobiliary system
can lead to imaging artifacts and interference with diagnosis. The present study investigated thetransport mechanisms of 99m Tc-TF and attempted to apply competitive inhibition using a specificinhibitor to reduce 99m Tc-TF hepatic accumulation. In this in vitro study, 99m Tc-TF was incubated in
HEK293 cells expressing human organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3,
OATP2B1, organic anion transporter 2 (OAT2), organic cation transporter 1 (OCT1), OCT2, andNa+ -taurocholate cotransporting polypeptide with or without each specific inhibitor to evaluate
the contribution of each transporter to 99m Tc-TF transportation. In vivo studies, dynamic planar
imaging, and single photon emission computed tomography (SPECT) experiments with rats were
performed to observe alterations to 99m Tc-TF pharmacokinetics using cimetidine (CMT) as an OCT1
inhibitor. Time–activity curves in the liver and heart were acquired from dynamic data, and the
99m Tc-TF uptake ratio was calculated from SPECT. From the in vitro study, 99m Tc-TF was found to betransported by OCT1 and OCT2. When CMT-preloaded rats and control rats were compared, thehepatic accumulation of the 99m Tc-TF was reduced, and the time to peak heart count shifted to an
earlier stage. The hepatic accumulation of 99m Tc-TF was markedly suppressed, and the heart-to-liver
ratio increased 1.6-fold. The pharmacokinetics of 99m Tc-TF were greatly changed by OCT1 inhibitor.
Even in humans, the administration of OCT1 inhibitor before cardiac SPECT examination may reduce
99m Tc-TF hepatic accumulation and contribute to the suppression of artifacts and the improvementof SPECT image quality.
Keywords: 99m Tc-tetrofosmin; small-animal SPECT/CT; hepatic uptake; transport mechanism; OCT1

iations.

1. Introduction
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

Coronary artery diseases are the leading cause of death in many developed countries.
To decrease mortality and morbidity, signs need to be identified as early as possible. Single
photon emission computed tomography (SPECT) is a useful diagnostic modality for cardiac
disease because SPECT images can reflect cardiac function.
Radiotracers for cardiac SPECT, such as 201 TlCl, 99m Tc-2-methyoxyisobutylisonitrile
99m( Tc-MIBI), 123 I-beta-methyl iodophenyl-pentadecanoic acid (123 I-BMIPP), and 3-123 Imetaiodobenzylguanidine (123 I-MIBG), are in widespread clinical use [1–3]. In particular,4.0/).

Pharmaceutics 2021, 13, 1073. https://doi.org/10.3390/pharmaceutics13071073

https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 1073

2 of 899m Tc-tetrofosmin (99m Tc-TF) is a well-known radiotracer for the detection of myocardial

perfusion abnormalities and can quantify some cardiac functions [4].
Generally, radiotracer accumulation in a specific target organ is desirable, but mostradiotracers also accumulate in other organs. In fact, 99m Tc-TF is known to accumulate in
the hepatobiliary system depending on the dose of 99m Tc-TF injected, blood flow to theliver, and excretions from the liver [5]. High accumulations in the hepatobiliary system cancause imaging artifacts and interfere with diagnosis. Therefore, despite the application ofvarious ingenious methods in the clinical stage to avoid adverse impacts, more effective
methods are still required.
It is known that 99m Tc-TF is carried into cells by passive diffusion and membranepotential difference [6,7]. However, we speculated that drug transporters are also involvedin the hepatic uptake of 99m Tc-TF because some 99m Tc-labeled compounds are transported
via solute carrier (SLC) transporters and adenosine triphosphate-binding cassette (ABC)transporters in the liver [8]. In particular, P-glycoprotein (MDR1) and multidrug resistanceassociated protein (MRP)1, MRP2, and MRP3 in ABC transporters are involved in thetransport of 99m Tc-TF [9]. Previous studies have shown that 99m Tc-TF has an affinity
for ABC transporters in the efflux of 99m Tc-TF into bile [9], but no reports appear to haveclarified SLC transporters for the hepatic uptake of 99m Tc-TF. If such SLC transporters could
be identified, the hepatic accumulation of 99m Tc-TF could be expected to be dramaticallyreduced when SLC transporters were inhibited by the competitive inhibition of SLC
transporters. The aim of the present study was to improve the image quality of cardiac
perfusion SPECT by reducing the liver uptake of 99m Tc-TF.
2. Materials and Methods
2.1. Uptake Experiments with HEK293 Cells
Transporter affinity was measured using human embryonic kidney 293 (HEK293) cells
transiently expressing human organic anion transporting polypeptide 1B1 (OATP1B1),
OATP1B3, OATP2B1, organic anion transporter 2 (OAT2), organic cation transporter 1(OCT1), OCT2, Na+ -taurocholate cotransporting polypeptide (NTCP), and mock cells. All
cells were purchased from GenoMembrane Inc. (Kanagawa, Japan). Dulbecco’s modified
Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Wako (Osaka,
Japan) and Thermo Fisher Scientific K.K. (Yokohama, Japan), respectively. A 99 Mo-99m Tc
generator (925 MBq) and tetrofosmin kit were purchased from FUJIFILM Toyama Chemical
Co. (Tokyo, Japan) and Nihon Medi-Physics Co. (Chiba, Japan), respectively.
For the competitive inhibition assay, bromosulflein (BSP, a substrate specific to OATP),p-aminohippuric acid (PAH, a substrate specific to OAT), cimetidine (CMT, a substrate specific to OCT), and taurocholate hydrate (TC, a substrate specific to NTCP) were purchasedfrom Sigma Chemical (St. Louis, MO, USA).
The cells were cultured in DMEM supplemented with 10% (v/v) FBS, 100 U/mL
penicillin and 100 µg/mL streptomycin at 37 ◦ C with 5% CO2 . The uptake experimentwith HEK293 was performed according to the methods described by Kobayashi et al. [8,9].
Two days before the uptake experiment, HEK293 cells were prepared at 4 × 105 cells/wellin 12-well plastic plates. Cells were pre-incubated for 5 min using modified Hank’sbalanced salt solution (MHBS). After pre-incubation, cells were then incubated with 0.5 mLof MHBS containing 99m Tc-TF (150 kBq/well) for 5 min as a control condition. In thecompetitive inhibition experiment, cells were incubated with 99m Tc-TF and 1.0 mM ofinhibitors for 5 min under the same control condition. At the end of incubation, eachwell was rapidly washed with ice-cold MHBS. Cells were solubilized in 0.1 mL of 0.1 N
NaOH and mixed with an ASC-II Scintillation Cocktail (GE Healthcare UK, Little Chalfont,
UK). Radioactivity was measured using a liquid scintillation counter (LSC-5100; Hitachi,
Tokyo, Japan). The amount of protein in cells was quantified using the bicinchoninic acid
method [10].

Pharmaceutics 2021, 13, 1073

3 of 82.2. Dynamic Planar and SPECT Imaging
A total of 6 male Wistar rats (8 weeks old; body weight, 200–230 g) were purchasedfrom Japan SLC, Inc. (Shizuoka, Japan). Animal studies were performed in accordancewith the recommendations of the Fundamental Guidelines for Proper Conduct of Animal
Experiment and Related Activities in Academic Research Institution under the jurisdictionof the Ministry of Education, Culture, Sport, Science, and Technology. The Animal Careand Use Committee of Nagasaki University approved all of the experimental protocols(approval number: 1506171238-2, 17 June 2015). Before the experiments, the rats were
fasted for about 4 h.
All imaging studies were performed using Triumph combined PET/SPECT/CT systems (TriFoil Imaging, Chatsworth, CA, USA). Control rats were intravenously administered 500 µL of saline (Otsuka Pharmaceutical Co., Tokyo, Japan), and treated rats wereintravenously administered CMT (200 µg/500 µL) as the OCT1 inhibitor. Six minutes later,
99m Tc-TF (80 MBq/500 µL/60 s) was injected via the tail vein. Rats were anesthetized with
1.5% isoflurane, and dynamic planar imaging was performed for 4.5 min at 5 s/frame.
After dynamic planar acquisition, SPECT was performed. SPECT acquisitions were performed for 21 min with 64 views over 360◦ , 20 s/projection, using a 60 mm radius ofrotation, starting 6 min after 99m Tc-TF injection. After SPECT, CT was performed foranatomical reference.
2.3. Image Analysis
Dynamic planar images were analyzed using VivoQuant 2.3 (INVICRO, Boston, MA,
USA). Regions of interest (ROI) were placed over the liver and heart. Time–activity curves
were calculated from ROI data and corrected by injected dose.
SPECT data were reconstructed using a 3D-maximum-likelihood expectation maximization algorithm (50 iterations). CT and SPECT data were processed and analyzed using
OsiriX MD (Pixmeo, Geneva, Switzerland). ROIs were placed over the liver and heart toobtain the total uptake of 99m Tc-TF. Heart-to-liver ratios were calculated.
2.4. Statistical Analysis
All results are given as the mean of least four experiments, expressed as mean ± SD.
Data were analyzed using the t-test or paired t-test, and values of p < 0.05 were considered
statistically significant. All statistical analyses were performed using Prism version 7.0
software (Graphpad Software Inc., San Diego, CA, USA).
3. Results
Table 1 and Figure 1 indicate 99m Tc-TF accumulation levels in HEK293 cells. No
significant differences were apparent between the control group and the inhibition groupin OATP1B1, OATP1B3, OATP2B1, OAT2, or NTCP. However, for OCT1 and OCT2, theaccumulated amount of 99m Tc-TF was decreased by competitive inhibition (p < 0.05).
Thus, OCT1 and OCT2 were speculated to be involved in the intracellular transportationof 99m Tc-TF.
Table 1. Details from Figure 1.
Control(%ID/mg Protein)

Inhibition(%ID/mg Protein)Mock

148.6 ± 22.0

143.6 ± 20.6

OATP1B1

141.5 ± 42.1

146.5 ± 21.3

OATP1B3

142.8 ± 38.0

136.5 ± 25.4

OATP2B1

163.7 ± 13.0

157.1 ± 11.4

OAT2

153.2 ± 18.3

150.9 ± 13.9

OCT1

204.3 ± 11.6

148.3 ± 12.8

OCT2

193.1 ± 18.2

143.7 ± 26.6

NTCP

153.9 ± 18.8

152.2 ± 22.4

Pharmaceutics 2021, 13, 1073

4 of 8Figure 1. The uptake of 99m Tc-TF in human HEK 239 cells expressing SLC transporter. The uptake of
99m Tc-TF was significantly decreased by the inhibitors of OCT1 and OCT2. ** p < 0.01 vs. control
condition, Student’s t-test.

OCT1 inhibition also affected the in vivo pharmacokinetics of 99m Tc-TF. As shown in
Figure 2, the liver counts of 99m Tc-TF reached a steady state in about 1 min after injection. In
control rats, steady-state values were higher than in CMT-treated rats. On the other hand,
the heart counts of 99m Tc-TF were markedly lower than those in the liver, and no difference
was identified between control rats and CMT-treated rats. However, in CMT-treated rats,
the time to peak heart count tended to shift to an earlier stage.

Figure 2. The time–activity curve of radioactivity in the liver and heart acquired by dynamic planar
imaging. Data acquisition was performed for 4.5 min at 5 s/frame. Solid line: control rats. Dotted
line: CMT-treated rats.

Figure 3 shows SPECT images in maximum intensity projection (MIP) mode. The
cardiac uptake levels of 99m Tc-TF did not differ between the control rats and the CMTtreated rats. However, the tendency toward hepatic uptake levels differed markedly. In thecontrol rats, 99m Tc-TF showed accumulation at a high level throughout the whole liver. On
the other hand, the hepatic uptake of 99m Tc-TF was suppressed by CMT preloading. The
heart-to-liver ratio increased 1.6-fold by the administration of CMT (Figure 4).

Pharmaceutics 2021, 13, 1073

5 of 8Figure 3. The SPECT images of a control rat (A) and a CMT-treated rat (B) injected with 80 MBq of
99m Tc-TF. The accumulation of 99m Tc-TF is high in the liver, but hepatic accumulation is markedly
decreased in CMT-treated rat liver and the boundary between the heart and the liver is clear.

Figure 4. The ratio of 99m Tc-TF uptake in the heart and the liver. The heart-to-liver ratio is calculatedas: (radioactive count for the whole heart)/(radioactive count for the whole liver). ** p < 0.01 vs.
control, Student’s t-test.

4. Discussion
In the present study, two main points merit attention. The first point is that the transport mechanism of 99m Tc-TF has been elucidated. Liver accumulation can be suppressed byinhibiting the uptake of 99m Tc-TF via OCT1. The second point is that this method appears
to offer a high possibility of clinical application, given that CMT is an inexpensive andfamiliar medicine that can be administered as an OCT1 inhibitor.
One of the advantages of cardiac SPECT is its capability to evaluate myocardial perfusion. The under- and overestimation of myocardial perfusion can lead to the misdiagnosisof heart diseases, especially for ischemic heart disease directly. Radiotracers for cardiac
perfusion diagnosis using 99m Tc tend to accumulate in the liver. Excessive accumulationin the liver adversely affects the evaluation and diagnosis of the left ventricular inferiorwall in contact with the liver. Methods to suppress accumulation in the liver are thus
necessary. To maintain the diagnostic performance of cardiac SPECT, various methods
have been reported. For example, Hara reported that the quality of cardiac SPECT imagesof the inferior wall improved by drinking soda water before SPECT with the expansionof the gastric wall and by avoiding the reflux of bile containing high radioactivity [11],Pharmaceutics 2021, 13, 1073

6 of 8

while Cherng reported that the liver excretion of 99m Tc-TF is accelerated by drinking lemon
juice [12]. Some research has been conducted to promote the excretion of radiotracers
from the liver and to improve the image quality of cardiac SPECT by eating fatty meals,
drinking milk, and so on [13]. However, those are not ideal methods, because there are
drawbacks, such as susceptibility to individual differences and the difficulty of global
standardization. By provoking the alteration of the pharmacokinetics of 99m Tc-TF withdrugs, not with food or drink, dosage per body weight can be precisely specified alongwith the timing of administration. In addition, with these methods, the preparation offoods or drinks was based on the idea of promoting the excretion of 99m Tc-TF accumulationin the liver. However, the rate of excretion is susceptible to individual differences andphysical conditions. Our research group therefore focused on the transporter mechanismin the liver and methods to block the hepatic uptake of 99m Tc-TF.
First, our research group attempted to identify the transporters that carry 99m Tc-TF intoliver cells. From the in vitro results, OCT1 and OCT2 are revealed to be strongly involved in
the intracellular transport of 99m Tc-TF. OCT1 is therefore known to be primarily expressedon the sinusoidal membrane of hepatocytes, and also on the basolateral membrane oftissues such as the heart, small intestinal enterocytes, renal proximal tubular cells, and soon [14,15]. On the other hand, OCT2 is not expressed in the liver [14–16]. We thus assumed
that the uptake of 99m Tc-TF into hepatocytes would be more affected than that into theheart when OCT1 is inhibited.
CMT is known to show high affinity for OCT1 and OCT2 [17–19], is low in toxicity, andhas a long history of clinical usage for the treatment of peptic ulcers. The CMT concentration
was determined by reference to blood CMT level in humans and the difference in metabolic
rates between humans and rats.
Dynamic planar imaging acquisition was conducted to track the pharmacokinetics of
99m Tc-TF at the early stage of administration. Despite the almost identical rates of increasein time–activity curves, the liver counts of the rats treated with CMT at a steady state weredecreased by about 20% compared to those of the control rats. This is because the uptakeof 99m Tc-TF from blood into the liver via OCT1 was inhibited by CMT. Accordingly, theamount of 99m Tc-TF trapped in the liver decreased, and the relative 99m Tc-TF inflow into
the heart immediately increased after administration. For these reasons, the timing of peakheart count was inferred to shift to an earlier stage. In clinical practice, myocardial perfusion
SPECT image acquisition using 99m Tc-TF is started 1 h after radiotracer administration inorder to wait for the reduction in the liver accumulation level of 99m Tc-TF. On the other
hand, Figure 2 indicated that the liver accumulation level of 99m Tc-TF was kept low by
preloading with CMT. The present results suggest the possibility of reducing the timerequired for myocardial perfusion SPECT inspection using 99m Tc-TF.
The alteration of the 99m Tc-TF pharmacokinetics caused by CMT is also reflected in
the SPECT images. The results of SPECT imaging show improvements in image quality,with obvious decreases in intrahepatic accumulation, and the clarification of the boundary
between the inferior cardiac wall and the liver. Figure 4 indicates not only the uptake
ratio of 99m Tc-TF, but also contrast between the heart and liver on SPECT imaging. The
experimental results show that the heart–liver contrast was significantly improved bypreloading with CMT. Of course, CMT is predicted to also inhibit OCT1 expressed inother organs. However, the effects of OCT1 inhibition on other organs are inferred to be
negligible from the results of dynamic planar imaging and the SPECT imaging studies.
The expression level and affinity to the substrates of OCT1 differ between species.
A difference in the transport activity of OCT1 is also seen between species. However, nomarked difference in substrate selectivity has been reported by species [20,21]. According
to Wang et al., OCT1 is expressed at 5-fold higher levels in the human liver as compared to
the rat liver [22]. More drastic effects would presumably be obtained if our method were tobe applied to humans.
In addition, the present study points out the important issue of drug combinations.
As stated already, CMT is used as a medication for reducing acid levels in the stomach.

Pharmaceutics 2021, 13, 1073

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Image quality would differ between patients already medicated with and without CMT.
The interpretation of those images could thus be influenced by information on concomitant
medications. Medical staff need to be aware of the influence of medications taken on a daily
basis and need to explain such issues to patients when planning cardiac SPECT examinations.
5. Limitations
All findings obtained in the present study were based on the results of in vitro experiments and animal experiments. The relevance of these findings to humans needs to be validated with appropriate clinical research. In addition, there is no certainty that the methodof suppressing liver accumulation by OCT1 inhibition can also applied to 99m Tc-MIBI.
6. Conclusions
In the liver, OCT1 is strongly involved in the intracellular transport of 99m Tc-TF. The
administration of OCT1 inhibitors can reduce the hepatic accumulation of 99m Tc-TF andimprove image quality for cardiac SPECT.
Author Contributions: Conceptualization, K.K. and K.N.; Methodology, K.N., I.T., and M.K.(Masato
Kobayashi); Investigation, K.N., M.K.(Minori Kikuchi), Y.M., and A.M.; Writing—original draft
preparation, K.N.; Writing—review and editing, K.N., M.K.(Masato Kobayashi), and T.K. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was supported in part by the program of the network-type joint Usage/ResearchCenter for Radiation Disaster Medical Science of Hiroshima University, Nagasaki University, andFukushima Medical University.
Institutional Review Board Statement: Animal studies were performed in accordance with therecommendations in the Fundamental Guidelines for Proper Conduct of Animal Experiment andRelated Activities in Academic Research Institution under the jurisdiction of the Ministry of Education, Culture, Sport, Science, and Technology. The Animal Care and Use Committee of Nagasaki
University approved all experimental protocols (approval number:1506171238-2).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors are grateful for the experimental support provided by Yuya Okui,
Kotaro Takahashi, Yutaro Mokudai, and Kohei Tsujino.
Conflicts of Interest: The authors declare no conflict of interest.

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